1. Field of the Invention
The present invention relates to a device and method for electroelution and also for dialysis, in particular such a device that is disposable. This invention more specifically relates to a device and method for the isolation of macromolecules, including proteins and nucleic acids, from a gel to a suitable solution within the device and for optionally further dialysing such macromolecules while still in the same device.
2. Background (Prior Art)
Agarose or polyacrylamide gel electrophoresis has been an essential and very powerful method for the purification or analyzing of proteins and nucleic acids in micro scale biochemical studies. Running macromolecules in such gel matrices plays a major role in molecular biology. The composition of the gel matrices may be chosen such as to enable separation of almost any macromolecule from a large pool comprising many different macromolecules allowing to serve as a tool for separate one molecule of interest. Depending upon various physical/chemical characteristics, a sample comprising different-sized macromolecules migrates through the electrical field at a particular velocity. After electrophoresis, the gel is removed from the electrophoresis chamber, if needed, stained with reagents specific for proteins and/or nucleic acids, destained with organic solvent mixtures and photographed. Whereas electrophoretic separation of macromolecules is an established technique, the elution of macromolecules from the gel has hitherto represented a difficult and generally non-reproducible procedure. Harvesting such macromolecules is potentially of commercial value because of their applications in science and medicine. The major problem is at recovering or extracting those macromolecules in high yields for down stream protocols. Examples of such down stream protocols include:- (a) Using a DNA fragment extracted from an agarose or polyacrylamide gel for constructing a new plasmide; (b) Separating a target macromolecule from contaminant molecules, for example double strand RNA (dsRNA) from single strand RNA, for using the dsRNA in activation of PKR; (c) Extraction of a protein from a polyacrylamide gel for use as antigen in vaccination; (d) Extraction of DNA or proteins for sequencing. In particular, recovery or extraction of macromolecules from agarose or polyacrylamide in high yields is a major problem. This problem becomes more severe as the size of the molecule of interest is increased or the percentage concentration of the gel of the separating matrix is high. Over the last decade or so, various attempts have been made to improve the yields of the recovering of macromolecules from gels.
Perhaps the simplest procedure for the elution of macromolecules involves a dialysis membrane. In one method, the membrane is in the form of a tubular skin that is closed at both ends after inserting the gel containing the sample. While the method represents an improvement in yield, it requires special skill to handle the sample, as well as to tie or clamp one end of the tube to form a sack and then the other end. Furthermore leakage and the presence of air bubbles interfere with the electric field.
Many research laboratory use an elution protocol to recover DNA or RNA from polyacrylamide gels which is a time consuming protocol with two major drawbacks, low yields (10–20% depend on the size of the eluted molecule) and sensitivity of the molecules to low contamination like DNase, RNase or Protases in the elution solution.
A similar approach has been taken to elute nucleic acids from agarose gels. Here, agarose gel is melted by heating to 65° C. The mixture is extracted with phenol and the samples eluted. As expected, recoveries are usually low with this procedure. In addition, phenol is a highly toxic and biohazardous substance. Since diethylaminoethyl (DEAE) cellulose binds deoxyribonucleic acid (DNA), it has been employed to elute DNA from gels. The procedure involves i) electrophoretic transfer of DNA from gels to DEAE-paper. ii) alternatively, DEAE-paper is inserted into slots immediately under each band, thus DNA is transferred electrophoretically. Although these procedures yield excellent recoveries, they are highly dependent on technique and the apparatus is expensive.
Decomposing, the gel with chemicals, followed by trapping the macromolecules on glass beads and their elution with salt solution is another method of elution. However, this method is dependent on buffer conditions and the solution that in use for the digestion contains significant contamination material in it and needs to wash out. More than that, this method recovers specially DNA or RNA from agarose and not from polyacrylamide gel, and the method cannot be used for the extraction of proteins.
To solve some of the problems represented above, another method was developed, using a container comprising a rigid tubular member having open ends that are sealed with membranes after the slice of the gel containing the sample molecule is inserted. Again, skill is required by the user, and the method is generally difficult and cumbersome. Furthermore, the device is reusable, requiring pretreatment before each use, leading to potential contamination problems and/or increasing complexity of use. Some new Electro-Eluter devices were developed that can process up to six samples simultaneously, but the devices represent high capital outlays. In many of those new Electro-Eluter devices, the sample is open to the environmental (ambient) air, which allows it to be easily contaminated. In order to reuse such devices, cleaning protocols need to be carefully followed to eliminate any contamination.
In 1985, Kartenbech introduced an electroelution apparatus (U.S. Pat. No. 4,552,640). This apparatus consists of an upper electrode in the upper chamber and the lower chamber to hold buffer solution and a lower electrode. The upper chamber is separated from the lower chamber by a septum, and the two chambers are connected by a connecting passage within the septum. The end of the lower chamber holds a dialysis membrane, wherein the electrophoretically eluted protein or polypeptide is collected. There are several disadvantages with this apparatus, including: i) since the volume of the lower chamber is large, it results in dilution of the sample, and ii) since the surface area of the dialysis membrane is large it results in non-specific adsorption of macromolecules resulting in very low recoveries.
In 1985, Walsh introduced an apparatus to elute nucleic acids (U.S. Pat. No. 4,545,888). This apparatus has features to introduce multiple copies of transfer chamber, filter discs to hold DEAE cellulose and negative electrode. Basically, in this procedure the sample is electrophoresed and collected on DEAE resin (held by a filter disc) at the bottom end of the lower chamber. Next, the filter disc is removed and DNA eluted from the resin employing standard elution protocols. This procedure requires an additional step involving the solution of nucleic acids from DEAE. Moreover, its application to elute proteins and polypeptides is uncertain.
In 1987, Burd introduced an electroelution method and apparatus (U.S. Pat. No. 4,699,706). This apparatus has features in which the electroeluted sample passes through a glass frit and is collected in a semipermeable membrane at the bottom end of the lower chamber. In this apparatus the dialysis membrane must be held in place by a retaining ring, a gasket and internal shoulders built in the equipment. There are several disadvantages with this equipment. For example, i) this is a rather complex setup and the success depends upon the technique used, ii) because the dialysis membrane is smaller then the diameter of the glass frit, it results in poor recovery, iii) use of dialysis membrane results in non-specific adsorption of macromolecules, which also contributes to low recovery, iv) there is no possibility of capping the columns to harvest the sample collected in the membrane, v) when the sample cup is removed, it leads to the disruption of the sample collected as it leaks through the filter disc and/or fluid held in the sleeve holding the cup.
In 1986, Clad introduced an apparatus for electroeluting macromolecules from gel (U.S. Pat. No. 4,608,147). This apparatus contains an upper chamber which holds a permeable membrane. (pore size about 0.2 micrometer) through which macromolecules can migrate downstream. The sample is collected in the lower chamber on top of an impermeable membrane having a molecular weight greater than 1000. Following elution, the polarity of the electric field is reversed for 10 to 15 seconds, so that the macromolecules adsorbed to the inner surface of the outer membrane are released from the membrane into the trap space. There are several disadvantages of this apparatus, including: i) the use of an impermeable membrane in the lower chamber results in dilution of sample, thus requiring further concentration, ii) because the sample is contaminated with the electrophoretic buffer, an additional step (e.g. dialysis) is required to remove such contaminants.
In 1990, Brautigam and Gorman introduced an electroelution apparatus (U.S. Pat. No. 4,964,961). This equipment consists of a tapered tube divided by a porous disc into an open upper section and a lower section which can be closed by a removable cap. The equipment has a dialysis membrane equal to the diameter of the removable cap and is affixed to it to close off the lower section. After electroelution, the upper section is closed. The sample is collected through the cup and dialysis membrane at the bottom end of the tube. Some disadvantages of this equipment include: i) the sample is contaminated and diluted with the electrophoretic buffer; accordingly, it requires dialysis and concentration, further adding to the time effort for such procedures, and ii) non-specific adsorption of sample to the dialysis membrane results in loss of recovery.
Dialysis is a molecular weight-based method of separating molecules through a semi-permeable membrane. The membrane by virtue of its composition and its porosity, allows molecules equal to or less than a particular molecular weight to cross the membrane. By using a membrane having a particular molecular weight cutoff the membrane will retain macromolecules higher than its molecular weight cutoff. On the other hand, it will allow the passage of molecules of a similar or lower molecular weight than the molecular weight cutoff of the membrane. The concentration gradient between the two sides of the dialysis membrane serves as the driving force of the process. There are four common application of dialysis membrane that are most often utilized by researchers in laboratory. 1) sample concentration, 2) sample desalting, 3) molecular separating and 4) exchanging buffer.
In a special application of dialysis, macromolecules recovered from a gel sample can be further filtered according to molecular weight. The most widely used dialysis method for such macromolecules in research laboratories uses a dialysis wherein the membrane is in the form of a tubular skin that is closed at both ends after inserting the gel sample, similar to one of the devices used for electroelution, described above. The sample solution is added to the interior of the dialysis membrane sack, which is then tied or clamp at the other end, which remained open. As with the parallel electroelution method, it requires special skill to handle the sample, as well as to tie or clamp one end of the tube to form a sack and then the other end. Furthermore leakage and the presence of air bubbles interfere with the dialysis process. Also, it is difficult to load and unload the sample from the sack because the sack is non-rigid. Many variations of this concept have been tried, albeit with little improvement.
U.S. Pat. No. 5,503,741 describes a device for dialysis of a liquid sample comprising a hermetically sealed vacant chamber formed by a gasket with dialysis membranes disposed on each side of the gasket without any supporting structure between the gasket and the membranes. The membranes are held in place over the gasket by means of inner surfaces of an external housing having windows. The gasket is impermeable to the sample being analyzed, and does not comprise an inlet opening. Rather, the gasket is penetrable by sharp means such as a needle, so that the same needs to be forcefully inserted through the gasket into the chamber in order to deliver a liquid sample thereinto. The gasket has a high memory function such that it is resealable to permit needle withdrawal without sample leakage. Thus, since the device does not have an opening into the chamber it may therefore only be used with liquid samples and not with samples contained in or carried by gels. It follows that such devices cannot be used at all for dialysis of samples contained in gels which comprise the substance of interest, and would teach away from being useful for electroelution processes conducted on a sample contained in gel. The sealed nature of the chamber is in fact a characterizing feature of this device, but this means that air needs to be evacuated from the chamber prior to injecting the liquid to be dialyzed, otherwise there is a buildup of pressure within the chamber which could serve to force some of the sample out, or could rupture the membranes. However, since the chamber is hermetically sealed, the removal of air has to be done by special means such as by using a needle to penetrate into the chamber.
The sealed aspect of the chamber is considered in this reference to prevent contamination with any substance in the air. However, since the needle has to force its way into the chamber via the gasket, any contaminants on the outside of the gasket will find their way into the chamber together with the needle.
This device has other shortcomings. Construction of the device requires the precise superposition of the membranes with respect to the gasket, on either side thereof, and then closure of two corresponding shells over the membranes and gasket. Since the membranes and gasket are substantially non-rigid components, this adds some complexity to the production process of the devices. The devices are of a non-standard shape and therefore not readily compatible with other laboratory equipment.
Other references of background interest include WO 94/01763, WO 96/26291, U.S. Pat. No. 5,200,073 and U.S. Pat. No. 4,576,702.